Clinical significance of EGF and EGFR expression changes in cryptorchid boys

Guo-Sheng YANG¹, Ren-Kang LU², Zhao-Dian CHEN¹

¹Department of Urology, First Affiliated Hospital, Medical School of Zhejiang University, Hangzhou 310003,China
²Institute of Reproductive Medicine, Wannan Medical College, Wuhu 241001, China

Asian J Androl 2002 Dec; 4: 255-258            

Keywords: cryptorchidism; epidermal growth factor; epidermal growth factor receptor

Abstract

Aim: To explore the changes of epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) expressions in cryptorchid children and their clinical significance. Methods: The level of serum EGF was measured by radioimmunoassay (RIA) and the expression of EGFR by immunohistochemistry. Results: (1) The level of serum EGF was significantly lower in cryptorchid children than in normal subjects at age group of 5-9 years (P<0.01) and 10-14 years (P<0.01), (2) The level of EGF was significantly lower in boys with impalpable testis than in those with extracanalicular and intracanalicular testes (P<0.01), (3) The serum EGF level increased significantly 6 months after orchiopexy (P<0.05), (4) The EGFR expression in testicular Leydig's cells was lower in 2~4 year-old boys than in those over 5 years (P<0.05) and (5) the EGFR expression was less positive in the impalpable group and the intracanalicular group than that of the extracanalicular group (P<0.01). Conclusion: The EGF and EGFR expressions may correlate with the age and the position of testes; orchiopexy improves the EGF and EGFR expressions in cryptorchid boys.

The incidence of cryptorchidism is increasing and currently around 1.8 % of boys are affected [1]. Common complications associated with cryptorchidism are an increased risk of infertility and testicular malignancy [2]. To date, the general consensus is that the failure of the testis to descend into the scrotum results in histological changes that may be detectable by two years old and become progressively worse with increasing age [3, 4]. It has been confirmed that testicular development and descent are controlled by the hypothalamo-pituitary-gonadal axis. It has been demonstrated [5, 6] that epidermal growth factor (EGF) reverses the antiandrogen-induced cryptorchidism and defective epididymal development; it is thus considered that the presence of paratesticular or testicular EGF controls the androgen-dependent epididymal and testicular development and/or descent. In this paper, the serum EGF changes and the testicular epidermal growth factor receptor (EGFR) expressions in cryptorchid boys were studied.

2 Materials and methods

2.1 Patients

From 1997 to 2000, 88 boys, aged 2 to 14 (7.1 3.5) years, diagnosed as cryptorchidism by palpation and ultrasonography (and later confirmed by surgery), were admitted to this Department. Hermaphroditism, chromosomal disease and retractile testis were excluded. Patients were divided into 3 age groups (2~4, 5~9 and 10~14 years) and further into 6 groups according to the location of the testis: the extracanalicular testis group [aged 2 to 14 (average 7.6 3.4) years], the intracanalicular testis group [2 to 14 (6.9 3.5) years], the impalpable testis group [2 to 9 (5.1 2.1) years], the unilateral cryptorchidism group [2 to 14 (7.3 3.7) years] and the bilateral cryptorchidism group [2 to 10 (6.7 3.3) years]. All the 26 cases [2 to 14 (6.8 3.6) years] underwent orchidopexy were reexamined 6 months after orchido-pexy. With informed consent, the serum EGF levels of 53 healthy boys [2 to 14 (7.3 3.4) years] were examined as the controls.

2.2 Measurement of serum EGF

Two to three mL blood was withdrawn from all the boys' forearm vein at 7 am. to 9 am.. Sera were prepared and kept at -25 before assaying. The concentration of EGF in serum was measured by the radioimmunoassay procedure with the use of J 125 h EGF Reagent Kit for RIA (sensitivity threshold 0.08 ng/ml); the EGF standards (0.08-10.0 ng) were produced from lyophilized recombinant human epidermal growth factor (EGF Human Recombinant, Sigma) and diluted with Eagle MEM. The activity of the samples labeled with ¹²⁵ I was measured with I'-radiation counter, Gamma Automat SN-682.

Testicular biopsy was performed on all undescended testes by puncture and the tissues were frozen on dry ice and embedded in OCT compound. They were stored at -70 until processed for immunohistochemistry. Specimens were cut into 5 mm sections and a frozen section aid kit was used. Sections were fixed in 10 % buffered formalin for 7 minutes, washed in phosphate buffered saline (pH 7.4) and treated with methanolic peroxide followed by acetone at -25 to block endogenous peroxidases. Immunohistological staining was performed using a rabbit polyclonal anti-epidermal growth factor receptor antibody. Primary antibody was diluted 1 to 500 in 3 % rat sera and incubations were performed overnight at 4 . Adjacent control sections were incubated with 3 % rat sera alone. Secondary antibody and peroxidase reagents were diluted 1 to 200 in 3 % rat sera and incubated for 20 minutes. Diaminobenzadine tetrachloride was used as the chromagen. Under a light microscope, the number of positive (brown stained) cells per high-field was assessed and five fields in each slide were examined. The expression of EGFR was regarded as positive when at least 25 % of cells stained positively.

Data were expressed in mean SD. Statistical analysis was performed using Student's t test and chi-square tests. Results were considered significant at P<0.05.

3 Results

3.1 Concentration of EGF in serum

The serum concentrations of EGF in cryptorchid boys were significantly lower than those in the controls (P<0.01). In 5~9 and 10~14 years old cryptorchid boys, the serum EGF concentration was significantly lower than that in the corresponding normal boys, but in 2~4 years old cryptorchid boys, it was not significantly different from that in the normal boys (P>0.05). Six months after orchidopexy, the serum EGF concentration increased significantly (P<0.05) in 26 boys (Table 1~4).

Table 1. Preoperative serum EGF level in cryptorchid and control boys. Student's t-test, ^bP<0.05,^cP<0.01, compared with controls.

Table 2. Preoperative serum EGF level in uni- and bilateral cryptorchid boys. Student t-test.

Table 3. Preoperative serum EGF level in cryptorchid boys with different testis locations. Student's t-test, P<0.05.

Table 4. Serum levels of EGF before and after orchido-pexy. Student t-test, ^cP<0.01, compared with preoperative group.

Immunohistochemical results showed that the EGFR expression in Leydig cells was significantly less positive (P<0.05) in cryptorchid boys over 5 years old, especially in those with impalpable and intracanalicular testes, than in those aged 2~4 years (Table 5~7, Figures 1 and 2).

Table 5. EGFR expression in Leydig cells of cryptorchid testes at different ages. Chi-square test, ^bP<0.05, compared with 2~4 years group.

Table 6. EGFR expression in Leydig cells of unilateral and bilateral cryptorchid testes. Chi-square test, P<0.05, compared with unilateral cryptorchid testesgroup.

Table 7. EGFR expression in Leydig cells of cryptorchid testes at different locations. Chi-square test, P<0.01.

Figure 1. Positive EGFR immunohistochemical staining in Leydig cells from a 2-year-old cryptorchid boy (arrowhead) 40.

Figure 2. Less positive EGFR immunohistochemical staining in Leydig cells from a 7-year-old cryptorchid boy 40.

Epidermal growth factor is an androgen regulated mitogenetic growth factor that has been shown to regulate ectodermal and mesodermal cellular development [7]. The major source of EGF is from the submandibular gland and is increased by androgenic stimulation [5]. The EGFR, a transmembrane protein, possesses a glycosylated extracellular domain with ligand binding sites and an intracellular domain with tyrosine kinase activity and autophosphorylation sites near the carboxyl terminus [8]. EGF apparently exerts its effects by binding to and activating the EGFR. After binding to EGF ligands, EGFR evokes subcellular protein tyrosine phosphory-lation, including rapid receptor autophosphorylation [9]. In many cell types, this results in numerous early and delayed responses, which leads to proliferation and differentiation [10].

Testicular development and descent is not only controlled by the hypothalamo-pituitary-gonadal axis, but also by the placental-testicular axis [11]. However, the exact mechanism for EGFR and EGF action on the descent and development of the testis is not clearly known. The following hypotheses have been suggested [5]: (1) a direct action on the fetal testes either independently or synergistically with placental gonadotropins to increase the fetal testicular androgen release, (2) a direct action on the target organs to guide their development, and (3) stimulation of the placenta, resulting in an increased release of placental gonadotropin, thereby enhancing fetal testicular androgen production. In rats, resection of the submandibular gland reduced the blood EGF level trace amounts and decreased the testicular sperm to 55 %, but the spermatocytes were increased by 20 %, indicating that spermatogenesis was arrested at the spermatocyte stage. It has been confirmed that EGFR and its ligand, EGF, do affect spermatogenesis [11]. Present study also showed that EGF has a number of biological effects, affecting cell proliferation and differentiation and protein and DNA synthesis.

As indicated that in undescended testis, histological changes are detectable by 2 years old and become progressively worse with increasing age and early orchiopexy at the age of 2 is beneficial to cryptorchid boys [3,4]. Our results support this view, as the serum EGF concentration was significantly lower in the older cryptorchid boys (5~14 years of age), but not in the younger ones (2~4 years) and six months after orchidopexy, the serum EGF was recovered to the normal level in the older boys.

References

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Correspondence to: Guo-Sheng YANG, Department of Urology, First Affiliated Hospital,Medical School of Zhejiang University, 79 Qingchun Road, Hangzhou 310003, China.

E-mail: ywy2008@163.com

Received 2002-03-05 Accepted 2002-11-12